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. 2014 May 15;289(28):19778–19788. doi: 10.1074/jbc.M114.573162

FIGURE 2.

FIGURE 2.

ORA1 is activated by 4-hydroxyphenylacetic acid, a contaminant of aged tyrosine. A, top row, cells were transfected with ORA1, and challenged with 100 μm of different stimuli as indicated. Calcium traces are averaged from 3 wells. Only aged tyrosine (first column), i.e. the tyrosine lot used in Fig. 1, and oxidized fresh tyrosine (third column) were active, but fresh tyrosine itself (second column) was not. Hydrogen peroxide itself, l-dopamine (l-DOPA), and l-phenylalanine (l-Phe) had no effect. Bottom row, cells transfected with empty vector showed no response to any of the stimuli. Shown are calcium traces averaged from 2 wells. Scaling: y axis = relative units, x axis = 1 min. B, a single active compound is detected in aged l-tyrosine. 100 μg of aged tyrosine was separated by HPLC fractionation using a water/methanol gradient with UV detection at 280 nm. Ten fractions corresponding to peak absorbance rates (labeled 1–10) were collected at the following time points during the run: 1, 3.4–5.6 min; 2, 6.0–6.9 min; 3, 7.5–8.2; 4, 8.2–9.2 min; 5, 9.2–10.2 min; 6, 10.7–11.5 min; 7, 13.7–14.9 min; 8, 15.5–17.4; 9, 20.6–23 min; 10, 25–29.5 min. y axis, absorbance units (AU); x axis, run time (min). Inset of B, chemical characterization of fraction 6 showed 4-hydroxyphenylacetic acid as the main compound. The carbon atoms of 4-hydroxyphenylacetic acid are labeled by arbitrary numbers to allow easy association with the following analytical, mass spectrometry, and NMR data that resulted in unambiguous identification: UV-visible (0.1% aqueous HCOOH)/MeOH; 6/4, v/v): λmax = 228, 276 nm; LC/TOF-MS: C8H8O3; LC/MS (ESI−): 151.1 (100; [M-H]−); 1H NMR (500 MHz; dimethyl sulfoxide-d6/MeOD; 9/1, v/v; COSY): δ 3.37 [s, 2H, H-C(5)], 6.67 [m, 2H, H-C(3,3′)], 7.02 [m, 2H, H-C(3,3′)]; 13C NMR (125 MHz; dimethyl sulfoxide-d6/MeOD; 9:1, v/v; DEPT-135, heteronuclear single quantum coherence, heteronuclear multiple bond correlation): δ 40.3 (CH2, C(5)), 114.8 (CH, C(3, 3′)), 125.6 (C, C(1)), 130.1 (CH, C(2, 2′)), 155.8 (C, C(4)), 173.2 (C, C(6)). C, each of the collected fractions was dried, redissolved, and used to stimulate ORA1-transfected HEK 293T cells. Only fraction 6 resulted in fluorescence changes. Last column, 100 μm unpurified aged l-tyrosine served as positive control (+con.). Cells transfected with empty vector showed no response to any fraction. Scaling: y axis, arbitrary units; x axis 9 min.